Thioflavin Research Articles

Thioflavin T (ThT) is a well-established fluorescence probe with selectivity for G-quadruplex (G4) structures. Over the past few years, G4 ligands have emerged as promising candidates for the development of antiparasitic agents. Building on this concept, we explored extending ThT's benzothiazole scaffold by introducing various 2-ethenyl aromatic and heteroaromatic moieties, aiming to enhance G4 binding affinity and potential therapeutic effect. A series of benzothiazolium derivatives were synthesized and evaluated for their antiproliferative and antiparasitic activity. Several 2-ethenyl benzothiazole derivatives showed submicromolar activity against Leishmania spp. and Trypanosoma brucei parasites, with up to 200-fold selectivity over MRC-5 human lung fibroblasts. Notably, compound 2b demonstrated remarkable potency, with an IC50 of 0.48 nM and a selectivity index of 46,151 against Leishmania major amastigotes, and an IC50 of 0.019 nM and a selectivity index of 79,206 against T. brucei. In fact, compound 2b demonstrated superior efficacy and selectivity in comparison to the clinically used drugs suramin, fexinidazole, miltefosine, and amphotericin B. Biophysical studies revealed that all tested derivatives exhibited significant G4 stabilization, surpassing ThT. Location of compound 2b inside the nucleus and the kinetoplast, as well as partially in the mitochondria, opens up the possibility of 2b acting against the parasite through binding to G4.

Molecular mechanisms of Lobeline-mediated inhibition of lysozyme amyloidogenesis: A synergistic approach using biophysical and cheminformatics techniques.

Background: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles, which synergistically accelerate disease progression. Since Aβ plaques and tau tangles are key factors in the development of AD, dual-targeting of Aβ and tau aggregation represents a promising therapeutic strategy. Amodiaquine (AQ), a quinoline-based antimalarial, has recently attracted attention for its ability to suppress protein aggregation. However, direct effects of AQ on both Aβ and tau aggregation remain unclear. Methods: The effects of AQ on the aggregation and dissociation of Aβ and tau were examined using a thioflavin T (ThT) assays. Molecular docking and molecular dynamics (MD) simulations were performed to examine binding characteristics and structural interactions. The effects of AQ on the expression of pro-inflammatory cytokines induced by Aβ and tau aggregation in BV2 microglial cells were analyzed by qRT-PCR. Results: ThT assay demonstrated a dose-dependent dual effect of AQ on Aβ, where 25 μM inhibited aggregation after 36 h, while 250 μM markedly accelerated it, reaching a plateau within 12 h. All concentrations of AQ promoted the disassembly of mature Aβ fibrils within 12 h. Molecular docking revealed stronger binding of AQ to aggregated Aβ (−45.17 and −23.32 kcal/mol for pentameric 2BEG and hexameric 2NAO) than to monomeric Aβ (−4.81 and −7.29 kcal/mol for 1Z0Q and 2BEG). MD simulation suggested that AQ disrupted the cross-β-sheet interactions of Aβ aggregates. In the case of tau, ThT assay showed that all concentrations of AQ inhibited tau aggregation from 6 h, and 350 μM AQ promoted the disassembly of mature fibrils from 6 h. Molecular docking indicated stronger binding of AQ to aggregated tau (−27.95 and −12.13 kcal/mol for the pentameric and decameric 5O3L) than to monomeric tau (−3.05 kcal/mol for 8Q96). MD simulations revealed no major structural changes in the aggregates. In BV2 cells, 1 and 10 μM AQ significantly reduced Aβ and tau-induced TNF-α and IL-6 mRNA expressions. In APP-H4 cells, 10 μM AQ decreased the level of Aβ compared to the control. Conclusions: AQ modulates both Aβ and tau aggregation and attenuates neuroinflammation and reduces Aβ pathology, supporting its potential as a dual-target therapeutic candidate for AD.

The aggregation of amyloid beta-42 (Aβ42) into β-sheet-rich fibrillar structures is a critical pathogenic feature of Alzheimer’s disease (AD). Baicalein (BCN), a natural flavonoid, has been shown to inhibit aggregation in amyloidogenic proteins, including human islet amyloid polypeptide (hIAPP), which shares structural similarities with Aβ42. This study investigates the inhibitory and disaggregation effects of BCN on Aβ42 using biophysical assays and atomistic molecular dynamics (AT-MD) simulations. Thioflavin-T (ThT) fluorescence, circular dichroism (CD) spectroscopy, and fluorescence microscopy reveal that BCN significantly reduces fibril formation and induces disaggregation of preformed Aβ42 fibrils in a concentration-dependent manner. Dynamic light scattering (DLS) analysis further confirms that BCN stabilizes Aβ42 in its monomeric form, preventing the formation of larger aggregates. AT-MD simulations show that BCN interacts with the aggregation-prone region of Aβ42, specifically disrupting the Asp23-Lys28 salt bridge, which is crucial for β-sheet formation. The simulations also reveal that BCN promotes the formation of α-helical structures, reducing β-sheet content and hindering aggregation. Secondary structure analysis via DSSP plots confirms that BCN shifts Aβ42 towards less aggregation-prone conformational states. These results highlight BCN’s dual function in inhibiting both the formation and disaggregation of Aβ42 fibrils. This study provides mechanistic insights into BCN’s therapeutic potential for amyloid-related diseases, suggesting that it can effectively target the β-sheet spine structures common to multiple amyloidogenic proteins, offering a promising approach for mitigating AD progression.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21991-7.

BRCA1-associated protein-1 (BAP1) is a tumor suppressor protein that regulates DNA transcription through its deubiquitinase activity. Cancer-associated missense mutations within its ubiquitin C-terminal hydrolase (UCH) domain result in structural destabilization and aggregation, contributing to various malignancies such as malignant mesothelioma and uveal melanoma. In this study, we investigated the aggregation mechanisms of highly destabilized BAP1-UCH variants, including N78S, C91W, F81V, and G128R, using Thioflavin T (ThT) binding assays and AmyloFit analysis. Our results reveal that all BAP1-UCH variants follow a secondary nucleation-dominated aggregation model, exhibiting strong concentration dependence and significantly higher aggregation rates, which may be responsible for their impaired nuclear import, leading to increased cytosolic retention. These findings provide critical insights into how specific mutations in BAP1 drive aggregation and compromise its tumor-suppressing functions inside the nucleus, thereby contributing to cancer progression.

14-3-3ζ protein prevents formation of GSK3β-phosphorylated Tau protein fibrils.

Dual cascade signal amplification lights up G4 dimers for fluorescent detection of DNA repair enzyme FEN1.

Monitoring Rocuronium Bromide (RcBr), a neuromuscular blocker widely used in anesthesia, is clinically important yet analytically underexplored. We report a fluorescence-based sensor for RcBr using Thioflavin-T (ThT) aggregates templated by γ-sulfated cyclodextrin (γ-SCD). This competitive displacement assay exploits the high binding affinity of Rocuronium for γ-SCD to displace Thioflavin-T from supramolecular aggregates, resulting in a fluorescence turn-off response. The sensor exhibits submicromolar sensitivity (LOD ∼ 0.2 ± 0.02 μM), rapid response (<1 min), and high selectivity over biological interferents. Its functionality in diluted human serum demonstrates potential for possible clinical applications. This work presents a novel optical approach for RcBr sensing that may support future developments in neuromuscular blockade monitoring.

Alpha-synuclein (αS) is an intrinsically disordered protein (IDP) that can self-assemble into amyloid fibrils, undergoing a transition from disordered monomers to ordered β-sheet-rich fibrils. The amyloid state of αS is implicated in various synucleinopathies, most notably Parkinson's disease (PD), in which αS fibrils accumulate as insoluble Lewy body deposits. Colocalized with αS in Lewy bodies are elevated levels of metal ions including Zn2+. We find in vitro that Zn2+ accelerates aggregation of N-terminally acetylated αS, decreasing the t50 ca. 5-fold, as measured by thioflavin T (ThT) fluorescence. Strikingly, the extent of Zn2+ binding (native mass spectrometry; MS) and shifts of the monomeric αS conformational ensemble toward compaction, measured using ion mobility MS (IM-MS) at different αS:Zn2+ ratios, mirror precisely the accelerated aggregation kinetics. Chemical shift perturbations in Nuclear Magnetic Resonance (NMR) spectroscopy were investigated together with molecular dynamics (MD) to map the Zn2+ binding sites and subsequent effects on conformation under identical solution conditions to those used in IM-MS. Zn2+ is found to predominantly interact with negative residues in the C-terminal region of αS but also His50 in the N-terminal region. This promiscuity in interactions potentially guides compaction of the protein chain by bridging residues between the N- and C-terminal regions through Zn2+ ion co-ordination. This study provides insights into the early stages of amyloid assembly, correlating aggregation kinetics with structural compaction in monomeric αS and highlighting the capability of native IM-MS to resolve complex structural ensembles of a disordered protein.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that catalyzes the reversal of O6-alkylguanine lesions, with an essential role in tumor resistance to alkylating chemotherapeutic agents. Herein, we develop a "repaired and activated" DNAzyme-RCA circuit for label-free and one-pot detection of MGMT in cells and tissues. The presence of MGMT can catalyze the demethylation of O6MeG-caged circular DNAzyme probe (O6MeG-cDZ), restoring its catalytic activity to specifically cleave adenine ribonucleotide (rA)-bearing substrate probe to produce a trigger sequence with a 2',3'-cyclic phosphate at its 3'-end. Subsequently, the resulting trigger can serve as a primer to initiate rolling circle amplification (RCA) upon healing its 3'-end by T4 polynucleotide kinase (T4 PNK), generating a large number of long G-quadruplex sequences. The G-quadruplex sequences can incorporate with thioflavin T (ThT) to produce a dramatically amplified fluorescence signal. Notably, O6MeG-cDZ integrates both a DNAzyme sequence and an RCA template sequence to achieve cleavage-and-amplification detection, efficiently eliminating nonspecific amplification. This method enables one-pot, isothermal, and label-free detection of MGMT down to 8.17 × 10-9 ng/μL and even quantification of MGMT at the single-cell level. Moreover, it can be employed for screening of MGMT inhibitors and precise discrimination of MGMT expression in breast cancer tissues and healthy counterparts, providing a novel paradigm for DNA repair enzyme-related clinical diagnosis and drug discovery.

Amyloid-β (Aβ) fibrillation is a spontaneous, thermodynamic process governed by nucleation and elongation. While many studies have explored the ability of engineered nanomaterials (ENMs) to modulate Aβ fibrillation, such as inhibitors, promoters, and dual-modulators, the key physicochemical property of ENMs that determines this behavior remains unclear. In this study, we developed a comprehensive library of ENMs with well-controlled physicochemical properties, including surface charges, morphologies, and hydrophilicity, to systematically investigate their effects on Aβ40 fibrillation. We identified hydrophilicity as the primary determinant of ENM-mediated modulation, rather than surface charge or morphology. Thioflavin T (ThT) kinetics assays indicated that hydrophilic ENMs exhibited bidirectional modulation, both promoting and inhibiting fibrillation depending on concentration. This bidirectional effect results from a competition between accelerated nucleation and decelerated elongation. While hydrophobic ENMs exhibited only unidirectional inhibition from the initial nucleation phase, two-dimensional-NMR (2D-NMR) mechanism studies indicated that this difference resulted from specific interactions with Aβ40 residues. Hydrophilic ENMs targeted hydrophilic residues involved in elongation, including Arginine R5, Glycine G9, G25, G33, G37, and G38, Lysine K28, and Alanine A30, while hydrophobic ENMs bound to hydrophobic residues critical for nucleation, such as I31. These findings provide mechanistic insight into NP-peptide interactions and lay a foundation for the rational design of nanomaterials to modulate amyloid fibrillation.

Unprecedented binding of Thioflavin T with well-ordered spherical aggregates: A false positive?

Early insulin fibril detection: Insulin fibril research and TR structural transition detection with FRET-Probe.

Effect of pyrimethanil on aβ42 aggregation mechanisms revealed at single entity level and molecular dynamic simulations.

Uncamarins A-D, oxindole alkaloids from Philippine Uncaria longiflora and their anti-amyloidogenic properties.

Concentration-dependent structural transition of huntingtin protein in Huntington's disease.

The precise measurement of microRNAs (miRNAs) is essential for diagnosing newborn pneumonia. This paper presents a simple, sensitive and accurate fluorescence-based technique for miRNA identification, utilizing a λ-exonuclease (λ-Exo)-driven DNA walker and split G-quadruplex (split-G4)-facilitated signal amplification. In this biosensor, target miRNA initiates the DNA walker by unfolding the Walker-probe, hence perpetually facilitating the reassembly of split-G4. The reformed intact G4 structure is distinctly identified by the commercially accessible fluorescent dye thioflavin T (ThT), facilitating highly sensitive, label-free miRNA identification. Additionally, the DNA walking process is motivated by the λ-Exo, which endows the biosensor with a greatly elevated signal amplification efficiency. This method demonstrates low background noise and good dependability owing to its reliance on split-G4-generated signals. Furthermore, the technique has been effectively utilized on clinical specimens, indicating its capability for disease diagnosis.

Hindering tau fibrillization by disrupting transient precursor clusters.

Evaluation of dual-function molecules containing both Zn-Ionophore and aggregation inhibition moieties for mutant p53 protein reactivation.

Effects of rosmarinic acid on the fibrotic toxicity of amylin in a zebrafish model.